BNA PCR Clamp and mutant-specific Probes.
Identification of rare mutant DNA species within an excess wild type sample is difficult. Up to 90% of sequence variants in humans often differ in only a single DNA base. This difference in a single DNA building block is called single nucleotide polymorphisms (SNPs). Polymorphisms within an individual may cause cancer or other diseases. Successful detection is essential for the characterizing and early detection of point mutations present in cancer patients, genetic diseases as well as others.
Over the past two decades, much research has focused on improving the selectivity of PCR-based technologies for enhancing the detection of minority (mutant) alleles in clinical samples. Routine application in clinical and diagnostic settings requires that these techniques be accurate and cost-effective and require little effort to optimize, perform, and analyze. However, the use of oligonucleotides for targeting duplex DNA is limited because of poor binding to the target and low stability against nuclease degradation.
Bio-Synthesis's Bridged Nucleic Acid (BNA) mediated PCR clamping and mutant-specific probes are versatile and sensitive detection probes. BNA clamps can be used for the identification of single nucleotide changes in DNA molecules ( < 0.01% of the total DNA). BNA clamping specifically blocks amplication of a given DNA template, while allowing amplification of another template that differs by as little as one nucleotide. BNA enhanced oligonuncleotides have unique properties allowing BNA clamping. First, BNA/DNA interactions are generally 2-4 oC per base pair more stable than the corresponding DNA/DNA duplex. Shorter probes can be designed to address traditionally problematic target sequences, such as AT- or GC-rich regions, highly repetitive sequences or regions with difficult secondary structure. Shorter regions of homology in aligned sequences can also be targeted. BNA is compatible with real time PCR methods. As a result, BNA enhanced DNA/BNA PCR clamps can be used to compete with probes and primers for binding. They are very similar to the primers or probes they compete with, but are designed to perfectly match undesired PCR products or templates that should not be amplified. BNA mediated mutation-specific detection probe are simple, sensitive, and an ideal solution for the detection of minority mutant population in the diagnosis of infectious diseases.
The Impact of BNA Enhanced clamped-probe real-time PCR
- Significantly increased sensitivity compared to other modified analogs
- Superior detection from challenging clinical samples such as biofluids and FFPE
- Increased target specificity compared to LNA and PNA probes
- Enables detection of single nucleotide mismatches
- Compatible with nucleic acid detection system.
Design suggestions: The ratio of BNA modifications used in the clamping oligonucelotides varies based on the type of BNA-NC analogs used, the length of the clamping probe, GC%, and the kind of mutation to be detected, as well as the Tm value. In general more than 50-80 % of a 10-15 mer DNA oligonucelotide should be modified with BNAs. BNA's should be introduced at the positions where specificity and discrimination is needed (For example at the 3' end in allele specific PCR and in the SNP position in allele specific hybridization probes). A 2’OMe modified base is used at 5’end for exonuclease protection. BNA/DNA clamps are designed so that they are not extended during the PCR reaction. We suggest modifying the 3' end of the BNA oligonucleotide with a phosphate group or a dideoxy terminal modifier to inhibit primer extension.
- Ideal BNA length: 10-18
- Avoid stretches of more than 4 BNA bases BNA hybridizes very tightly when several consecutive residues are substituted with BNA bases.
- Each BNA bases increases the Tm by approximately 2-4 oC.
- Do not use blocks of BNA near the 3' end
- Tm of the primer pairs should be nearly equal
- BNA/DNA base ratio